Process for synthesizing protein using cell-free protein synthesis system

ABSTRACT

A method for producing a protein using a cell-free protein synthesis system comprising a detergent so that the protein can be synthesized without aggregation, is provided. The protein is a protein comprising a hydrophobic region in at least a portion thereof, for example, a membrane protein or its fragment (portion). And the detergent is a mild detergent which would not denature the protein, for example, a nonionic or amphoteric ionic detergent.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP02/04204 which has an Internationalfiling date of Apr. 26, 2002, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a method for producing a protein byusing a cell-free protein synthesis system, and, in particular, to amethod for producing an insoluble protein that is associated with orimplanted in a membrane in vivo, such as a receptor, withoutaggregation, as well as to a method for reconstituting a proteinsynthesized thereby.

BACKGROUND ART

A large-scale analysis of genome sequences of various organisms such ashuman has been studied, and its goal is almost achieved. It is a nexttask to elucidate functions of proteins, which are encoded in enormousnumbers of genes discovered by the sequence analysis. Findings obtainedby these protein functions are expected to be greatly useful for thedevelopment of new drugs. The analysis of the three dimensionalstructure of protein provides useful information for elucidating theprotein function or drug design. Hereafter, its importance will beincreasing and a high throughput analysis in accordance with a largescale analysis will be desirable.

A purified protein in milligram order is necessary for the analysis ofthe three dimensional structure of protein. Previously, a large-scalepreparation of protein was a bottle-neck for the three dimensionalstructural analysis, however, a desired protein can be easily preparedin large-scale, by the advanced gene cloning techniques using anexpression system such as that of a microorganism or a cultured cell atthe moment. Further, a cell-free protein synthesis system has beenimproved by various methods such as dialysis and the like, to obtain aprotein in milligram order for several hours. Thus, the high throughputanalysis of three dimensional structure of protein is coming true.

However, these methods are not always applicable to every kind ofproteins, and it is still difficult to prepare a protein that has ahydrophobic region, such as a membrane protein, in large amount. In theexpression system of cultured cell, a membrane protein is accumulated inthe cell membrane by the localization system of the host cell. Thus,when the expressed protein is purified from the cultured cell, a step toextract the protein from the membrane by using detergents is necessary.This step needs much expense in time and effort, and is not so efficientin the extraction. Some kinds of detergent often impair the intrinsicstructure and function of the protein. When a protein that has ahydrophobic region therein, such as a membrane protein is expressed inE. coli, the expressed protein often forms insoluble precipitation.Therefore, it is necessary to solubilize the precipitate by using astrong denaturating agent such as guanidine salts or urea, and torenaturate or refold the denatured protein to its intrinsic structure (afolding step) in the purification. These steps are also at much expensein time and effort, and have many problems such as the reprecipitationduring the folding step. These proteins also form an insolubleprecipitate in a cell-free protein synthesis system, so that the amountof the synthesized protein is not sufficient.

As described above, the problem to solubilize membrane proteins makes itdifficult to prepare a large amount of the proteins, and retards theanalysis of three dimensional structures of the proteins under thecurrent circumstances. Among membrane proteins, however, there are manyimportant proteins for the target of drug development such as areceptor, channel protein, transporter and the like. The structuralanalysis of these proteins is urgently necessary for the efficientdevelopment of the drug.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to synthesize ahydrophobic protein (a protein comprising a hydrophobic region) such asa membrane protein, in cell-free protein synthesis system withoutaggregation (insolubilization).

In view of the problems described above, the present inventors havestudied, using a cell-free protein synthesis system, the synthesis of aninsoluble protein, in particular, a highly hydrophobic protein such as areceptor, which is embedded in the membrane in vivo. The protein hasbeen synthesized without aggregation (insolubilization) by a simplemethod, that is, an addition of detergents and/or lipids. Thussynthesized protein is found to have biological functions with greatprobability, even though it is not in the native states in vivo, such asa membrane bound state, and also can be used for the structural andfunctional analysis of the protein. These findings have led to thecompletion of the present invention.

According to a first aspect of the present invention, there is provideda method for producing a protein using a cell-free protein synthesissystem comprising a detergent to synthesize the protein withoutaggregation. In a preferred embodiment of the present invention, saidprotein comprises a hydrophobic region in at least a portion thereof,for example, a whole or portion (a partial structure) of membraneprotein and the like.

In a further preferred embodiment of the present invention, saiddetergent is a mild detergent that does not denature the protein, forexample, a nonionic or amphoteric ionic detergent. To be more specific,said detergent is at least one selected from the group consisting ofdigitonin, polyoxyethylene alkylether (Brij series), polyoxyethylenesorbitan (Tween series), β-dodecylmaltoside, β-octylglucoside,β-nonylglucoside, β-heptylglucoside, β-octylthioglucoside, sucrosemono-decanoate, sucrosemonododecanoate, octyltetraoxyethylene,octylpentaoxyethylene, dodecyloctaoxyethylene,N,N-dimethyldecylamine-N-oxide, N,N-dimethyldodecylamine-N-oxide,N,N-dimethyldodecylammonio propanesulfonate, octyl(hydroxyethyl)sulfoxide, octanoyl-N-methylglucamide,nonanoyl-N-methylglucamide, decanoyl-N-methylglucamide, and(3-[(3-cholamidepropyl)-dimethylammonio]-l-propanesulfonate (CHAPS).

In one embodiment of the present invention, there is provided a methodfor producing a membrane protein without aggregation by comprising adetergent in a cell-free protein synthesis system using a bacterial cellextract. Said detergent is preferably digitonin in 0.1 to 2.0% by volumeand/or Briji35 in 0.01 to 0.5% by volume.

According to a second aspect of the present invention, there is provideda method for reconstituting a protein produced in a cell-free proteinsynthesis system comprising a nucleic acid template coding for at leasta portion of a membrane protein, a detergent, and a lipid, wherein saidprotein is reconstituted in a lipid bilayer by decreasing theconcentration of said detergent in said system simultaneously with theprotein synthesis or after a period therefrom.

In a preferred embodiment of the present invention, the step ofdecreasing the concentration of said detergent is performed by any oneor more methods selected from the group consisting of dialysis,dilution, filtration, centrifugation and addition of an adsorbent tosaid detergent.

Further, in one embodiment of the present invention, there is provided amethod for reconstituting a protein comprising the steps of: (a)synthesizing the protein in a cell-free protein synthesis systemcomprising a cell extract, a nucleic acid template coding for saidprotein, a detergent and a lipid; and (b) decreasing the concentrationof said detergent in the reaction mixture simultaneously with theprotein synthesis or after a period therefrom, wherein the synthesizedprotein has at least a part of its biological activity.

In a preferred embodiment of the present invention, said membraneprotein is a protein selected from the group consisting of a receptor, achannel protein, a transporter, and a membrane-bound enzyme, or aportion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic diagram to prepare the template DNA used forthe expression of neurotensin receptor (NTR) in cell-free proteinsynthesis system, by PCR method.

FIG. 2 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofneurotensin receptors (NTR), which were synthesized in cell-free proteinsynthesis system added with digitonin, by western blotting analysis.Insoluble fractions precipitated by centrifugation after synthesisreactions were applied in lanes 1 to 5, and the supernatant fractions ofthe centrifugation were applied in lanes 6 to 10. The amounts ofdigitonin added to the reaction mixture were 0% (lanes 1 and 6), 0.04%(lanes 2 and 7), 0.4% (lanes 3 and 8), 1% (lanes 4 and 9) and 0% withouttemplate DNAs (lanes 5 and 10), respectively. Lane M was the molecularweight marker (ECL protein molecular weight markers by AmershamPharmacia Biotech).

FIG. 3 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofneurotensin receptors (NTR), which were synthesized in cell-free proteinsynthesis system added with Brij35, by the western blotting analysis.Insoluble fractions precipitated by centrifugation after synthesisreactions were applied on lanes 1 to 5, and the supernatant fractions ofthe centrifugation were applied on lanes 6 to 10. The amounts of Brij35added to the reaction mixture were 0% (lanes 1 and 6), 0.01% (lanes 2and 7), 0.02% (lanes 3 and 8), 0.2% (lanes 4 and 9) and 0% withouttemplate DNAs (lanes 5 and 10), respectively. Lane M was the molecularweight marker (ECL protein molecular weight markers by AmershamPharmacia Biotech).

FIG. 4 shows the schematic diagram to prepare the template DNA used forthe expression of human β2-adrenergic receptor (ADRB2) in cell-freeprotein synthesis system, by PCR method.

FIG. 5 shows the result of SDS-Polyacrylamide Gel Electrophoresis ofhuman β2-adrenergic receptor (ADRB2) synthesized in cell-free proteinsystem comprising digitonin, and analyzed by autoradiography. Insolublefractions precipitated by centrifugation after synthesis reactions wereapplied on lanes 1 to 5, and the supernatant fractions after thecentrifugation were applied on lanes 6 to 10. The amount of digitoninadded to the reaction mixture were 0% (lanes 1 and 6), 0.04% (lanes 2and 7), 0.4% (lanes 3 and 8), 1% (lanes 4 and 9) and 0% without templateDNAs (lanes 5 and 10), respectively. Lane M was the molecular weightmarker (ECL protein molecular weight markers by Amersham PharmaciaBiotech).

FIG. 6 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofhuman β2-adrenergic receptor (ADRB2) synthesized in cell-free proteinsynthesis system comprising digitonin, by the western blotting analysis.Insoluble fractions precipitated by centrifugation after synthesisreactions were applied on lanes 1 to 5, and the supernatant fractions ofthe centrifugation were applied on lanes 6 to 10. The amounts ofdigitonin added to the reaction mixture were 0% (lanes 1 and 6), 0.04%(lanes 2 and 7), 0.4% (lanes 3 and 8), 1% (lanes 4 and 9) and 0% withouttemplate DNAs (lanes 5 and 10), respectively. Lane M was the molecularweight marker (ECL protein molecular weight markers by AmershamPharmacia Biotech).

FIG. 7 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofhuman β2-adrenergic receptor (ADRB2) synthesized in cell-free proteinsynthesis system comprising Brij35, by the western blotting analysis.Insoluble fractions precipitated by centrifugation after synthesisreactions were applied on lanes 1 to 5, and the supernatant fractions ofthe centrifugation were applied on lanes 6 to 10. The amounts of Brij35added to the reaction mixture were 0% (lanes 1 and 6), 0.01% (lanes 2and 7), 0.02% (lanes 3 and 8), 0.2% (lanes 4 and 9) and 0% withouttemplate DNAs (lanes 5 and 10), respectively. Lane M was the molecularweight marker (ECL protein molecular weight markers by AmershamPharmacia Biotech).

FIG. 8 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofhuman β2-adrenergic receptor (ADRB2) synthesized in cell-free proteinsynthesis system comprising β-dodecylmaltoside, NP-40, Tween 20 orTriton X-100, and analyzed by autoradiography. Samples synthesized byadding no detergent, 0.5% each of β-dodecylmaltoside, NP-40, Tween 20,Triton X-l00 were applied on lanes 1 to 5, respectively. P showsinsoluble fractions, and S shows supernatant fractions. Lane M was themolecular weight marker (ECL protein molecular weight markers byAmersham Pharmacia Biotech).

FIG. 9 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofhuman β2-adrenergic receptor (ADRB2) synthesized in cell-free proteinsynthesis system comprising β-dodecylmaltoside, NP-40, Tween 20 orTriton X-100, and analyzed by the western blotting analysis. Samplessynthesized by adding no detergent, 0.5% each of β-dodecylmaltoside,NP-40, Tween 20, Triton X-100 were applied on lanes 1 to 5,respectively. P shows insoluble fractions, and S shows supernatantfractions. Lane M was the molecular weight marker (ECL protein molecularweight markers by Amersham Pharmacia Biotech).

FIG. 10 shows the results of SDS-Polyacrylamide Gel Electrophoresis ofrespective purification steps of human β2-adrenergic receptor (ADRB2)reconstituted by the method shown in Example 3, and analyzed by (a)silver staining, and (b) the western blotting analysis. Samples ofpurification steps by Ni—NTA agarose column, and those by PD-10desalting column were applied on lanes 1 to 5 (1:crude sample, 2:flowthrough fraction, 3:wash out fraction, 4:eluted fraction with 300 mMimidazole, 5:eluted fraction with 500 mM imidazole), and lanes 6 to 8(6:flow through fraction, 7:fraction No.1, 8:fraction No.2),respectively.

FIG. 11 shows binding curves of the His6-β₂ protein reconstituted inExample 3 and human β2-adrenergic receptor (Sf9) used as a control toAlprenolol.

PREFERRED EMBODIMENTS OF THE INVENTION

[Cell-Free Protein Synthesis System]

In the present invention, the cell-free protein synthesis system is anin vitro protein synthesis system using a cell extract. The system maybe either a cell-free translation system for producing proteins onribosome through reading of information of mRNA, or a coupled systemcomprising a cell-free transcription system that produces mRNA using DNAas a template and a cell-free translation system that translates themRNA information into proteins. When DNA is used for a template, variouskinds of template DNA can be prepared simultaneously and rapidly, by theamplification reaction in vitro such as Polymerase Chain Reaction (PCR),without a complicated procedure of molecular cloning into a living cell,which was previously required.

As the above cell extract, eukaryotic or prokaryotic cell extractcontaining factors required for protein synthesis such as ribosome,tRNA, and the like can be used. Any eukaryotic or prokaryotic cell whichis known in the art can be used. For example, E. coli, thermophilicbacteria, wheat germ, rabbit reticulocyte, murine L-cell, Ehrlichascitic cancer cell, HeLa cell, CHO cell, and budding yeast can be used.Especially, E. coli cell extract (for example, E. coli S30 cell extractfraction) or Thermus thermophilus cell extract is desirable for the highyield. The E. coli S30 cell extract fraction can be prepared from E.coli A19 strain (rna, met), BL21 strain, BL21 star strain, BL21 codonplus strain, and the like in accordance with generally known methods(refer to Pratt, J. M. et al., Transcription and translation—a practicalapproach, (1984), pp. 179–209, Henes, B. D. and Higgins, S. J. eds., IRLPress, Oxford), or can be purchased from companies such as Promega orNovagen.

Either a concentrated extract of each of above cell extracts(hereinafter referred to as “concentrated cell extract”) or anon-concentrated one (hereinafter referred to as “crude cell extract”)can be used as a cell extract, however, a higher yield of synthesizedprotein can be achieved in the case of concentrated cell extract. Toprepare the concentrated cell extract, any method such asultrafiltration, dialysis, PEG precipitation, and the like can be used.

In addition to the crude cell extracts or concentrated cell extract (10to 90 weight %) such as E. coli S30 fraction, the cell-free proteinsynthesis system of the present invention may contain DNA or RNA (mRNAand the like) coding for target proteins, ATP (0.5 to 5 mM), GTP (0.05to 1.0 mM), CTP (0.05 to 1.0 mM), UTP (0.05 to 1.0 mM), buffersolutions, salts, amino acids, RNase inhibitors, antibacterial agents,RNA polymerase if necessary (in case where DNA is used as a template),and tRNA and the like. In addition, it can contain ATP regeneratingsystems, polyethyleneglycol (for example, PEG#8000), 3′, 5′-cAMP, folicacids (0.1 to 5 mM), reducing agents (for example, 1 to 10 mMdithiothreitol) and the like.

For the buffer solution, for example, buffer agent such as Hepes-KOH orTris-OAc can be used. For salts, acetate (for example, ammonium salts,magnesium salts and the like) or glutamate salts can be used. Forantibacterial agents, sodium azide or ampicillin can be used. In casewhere DNA is used for the template, RNA polymerase is added to thereaction system, and enzymes on the market such as T7 RNA polymerase canbe used.

In the present invention, a combination of 0.02 to 5 μg/μL of creatinekinase (CK) and 10 to 100 mM of creatine phosphate (CP) is preferablyused as the ATP regenerating system, but is not limited only to thissystem. Any material which is known on the prior art can be used. Inaddition to the combination described above, for example, a combinationof 1 to 20 mM of phosphoenolpyruvate (PEP) and 0.01 to 1 μg/μL ofpyruvate kinase (PK) also can be used. PK and CK are enzymes whichregenerate ADP to ATP, and require PEP and CP as the substraterespectively.

The cell-free protein synthesis system of the present invention can becarried out by a batch method, flow method, and any other techniquespreviously known, for example, those methods are ultrafiltration method,dialysis method, column chromatography method using a resin on whichtemplates for translation are immobilized (refer to Spirin, AS. et al.,Meth. In Enzymol. volume 217, pp. 123–142, 1993), and the like.

[Insoluble Proteins]

A protein to be synthesized in the present invention is a protein thatcomprises a highly hydrophobic region locally in the molecule (insolubleprotein), or a portion thereof. Examples of the protein are, inparticular, membrane proteins such as a receptor, a channel protein, atransporter, and a membrane-bound enzyme. To be more specifically, themembrane receptors are exemplified by ion channel-contained receptorssuch as glutamate receptors in brain, seven membrane-spanning typereceptors (aminergic receptors such as adrenergic and dopaminergic, andreceptors for bioactive peptides such as angiotensin and neuropeptides),adipose receptors such as prostaglandin receptors, peptide hormonereceptors such as ACTH and TSH receptor, and chemokine receptors. Thetransporters are exemplified by those transporting relatively smallmolecules such as glucose and amino acid, furthermore relatively largemolecules such as proteins and DNAs. As the membrane-bound enzyme, anumber of proteins such as G protein that participate in the signaltransduction into cells exist, and play important rolls concerning cellproliferation and malignant transformation. In addition to thesepreviously known membrane proteins, the protein include novel membraneproteins that have unknown functions.

In some cases, these insoluble proteins may be used to analyze thebiological functions and three dimensional structures surprisingly bycomplexing with detergents. For example, soluble fractions of mousebrain extracted with detergents were reported to be detected the bindingactivity with neurotensin (refer to Mazella, J. et al., J. Biol. Chem.263, p. 144–149, 1988). In addition, a photosynthetic reaction centercomplex of rhodobacter (Rhodopseudomonas viridis) has been crystallizedas a complex with detergents, and a crystal structure of the complex wassolved in a high resolution more than 3 Å (refer to Michel, H. et al.,J. Mol. Biol., 158, p. 567-, 1982; and Deisenhofer, J. et al., Nature916, p. 618- (1985)). Thus, membrane proteins are likely to bereconstructed to their original states in lipid bilayer, even thoughthey are crystallized under conditions of covering with substantialamount of detergents.

Therefore, the protein synthesized according to the method of thepresent invention without aggregation, and recovered from supernatant ofthe reaction mixture has a high possibility to exert biological functionsuch as ligand binding activities or signal transducing activities.

[Detergents]

The detergent to be used in the present invention is preferably selectedand used appropriately in connection with distinct kinds of proteins tobe synthesized. Any detergent known in the art, which does not denaturethe proteins can be used. Such detergents which are usually used, areroughly classified into a nonionic, anionic, and amphoteric ionicdetergent in connection with its electronic property. The nonionicdetergents are exemplified by digitonin, polyoxyethylene alkylether(Brij series), polyoxyethylene sorbitan (Tween series),β-dodecylmaltoside, β-octylglucoside, β-nonylglucoside,β-heptylglucoside, β-octylthioglucoside, sucrose mono-decanoate, sucrosemono-dodecanoate, octyltetraoxyethylene, octylpentaoxyethylene, anddodecyloctaoxyethylene. The anionic detergents are, for example,taurodeoxycholic acid and the like. The amphoteric ionic detergents areexemplified by N,N-dimethyldecylamine-N-oxide,N,N-dimethyldodecylamine-N-oxide, N,N-dimethyldodecylammoniopropanesulfonate, octyl (hydroxyethyl)sulfoxide,octanoyl-N-methylglucamide, nonanoyl-N-methylglucamide,decanoyl-N-methylglucamide, and (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

These detergents can be used as single species, or combine more than twospecies. The working concentration of these detergents is preferablyadjusted depending on the kinds of target proteins, however, theconcentration of usually used is preferably 1 to 50 times of criticalmicelle concentration (CMC) of the detergent, more preferably 3 to 10times thereof. For example, in case where the nonionic detergents suchas digitonin and Briji35 are used as a detergent, the concentration ofdigitonin is preferably 0.1 to 2.0% by volume, more preferably 0.4 to1.5% by volume. The concentration of Brij35 is preferably 0.01 to 0.5%by volume, more preferably 0.02 to 0.2% by volume.

The above detergent is preferably a mild detergent that does notdenature the protein. Detergents that have a strong potency of proteindenaturation such as sodium dodecyl sulfate (SDS) is likely to denaturethe synthesized protein. In addition, these detergents are notappropriate for the method of the present invention due to thepossibility to denature the enzyme protein consisting of the cell-freesystem and inhibit the protein synthetic activity thereof.

Further, in case where the detergent by itself is not sufficient tosustain the protein structure in aqueous solution and to prevent theaggregation, it is capable of preventing the aggregation for the proteinto be coexisted with amphiphilic substances such as heptane-1,2,3-trioland octane-1,2,3-triol, which are smaller than the above detergent orlipid, and with polar substances such as triethylamine ammonium andphenylalanine.

[Detection of Synthesized Proteins and Reconstitution Thereof]

The protein synthesized in the cell-free protein synthesis systemaccording to the method of the present invention is detected in thesupernatant after normal centrifugation of the reaction mixture, withoutaggregation (precipitation) in the reaction mixture by complexing withthe detergent, as concretely shown in the following examples. Thus, theprotein in the solution can be used to analyze the function and thestructure by NMR, further to crystallize the protein from the solutionto analyze the X-ray crystal structure.

It is further preferable to reconstitute the protein synthesized in thecell-free protein synthesis system with artificial membranes, liposomesor the like, in order to analyze the structure and function of themembrane protein or the like in vivo more precisely. The synthesizedprotein is reconstituted in a lipid membrane by decreasing theconcentration of detergent simultaneously with the protein synthesis orafter a certain period from the protein synthesis, in a cell-freeprotein synthesis system supplemented with the detergent and lipid. Asused herein, the term “reconstitution” refers to the construction of thesystem that is homologous to the state in vivo by embedding at least aportion of the synthesized membrane protein in artificial membranes orliposomes which are constituted of lipid bilayer or multi-layer. Thelipids which can be used for this method comprises simple lipid such asacylglycerol (neutral lipid), cholesterol ester and the like, andcomplex lipid such as phospholipid and glycolipid. Phospholipds includephosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol(PG), plasmalogen, sphingomyelin, ceramide ciliatin, and derivativesthereof, while glycolipids are exemplified by cerebroside, globoside,ganglioside and the like, which are generically calledsphingoglycolipid. One or more of these lipids may be used incombination, in an amount of usually 0.1 to 10 mM, which should beproperly adjusted depending on the used lipid.

The membrane protein synthesized in the presence of these lipids isreconstituted by incorporating therein, when the lipid bilayer ormulti-layer is formed by decreasing the concentration of detergent.Methods for decreasing the concentration of detergent are exemplified bydialysis, dilution, and addition of an adsorbent to the detergent.

When the membrane protein that has been reconstituted in the lipidmembrane is purified, it is possible to recover these complex byfiltration, centrifugal separation or the like, and further tosolubilize the protein once more by adding the detergent to purify thetarget protein. Example 3 as described below, shows that the membraneprotein thus purified and reconstituted in the lipid membrane has aligand binding activity as human β2-adrenergic receptor (ADRB2). Theligand binding activity is assayed by measuring a specific binding(incorporation) of radioisotope of an antagonist, which binds orinhibits competitively with the protein in the various molar ratio ofthe radio-labeled or non-labeled antagonist of β-adrenergic receptorsuch as Alprenolol. According to the results of FIG. 11, thereconstituted β-adrenergic receptor is recognized to have a biologicalfunction to bind specifically with its ligand, because the radio-labeledamount that binds to β-adrenergic receptor is decreasing when theconcentration of non-labeled antagonist is increased.

Further, in case where the cell-free system is constituted of dialysismethod, it is possible to reconstitute the synthesized protein in thelipid layer by adding the detergent and lipid into the internaldialysate of the synthetic reaction, and removing the detergent from theexternal dialysate simultaneously with the protein synthesis or after aperiod from the protein synthesis, to optimize the concentration ofdetergent in accordance with the rate of protein synthesis.

EXAMPLES

The present invention is explained in more detail by reference to thefollowing examples using cDNA fragments coding for a rat neurotensinreceptor (NTR) and a human β2-adrenergic receptor (ADRB2). Theseexamples, however, do not restrict the scope of the present invention.Incidentally, “%” means “volume %” unless otherwise specified in thespecification.

Example 1

Synthesis of a Rat Neurotensin Receptor (NTR)

NTR is a member of G protein coupled receptors, a seven-spanningmembrane protein. By binding with neurotensin as a ligand, the receptorexhibits a function to activate phospholipase C through G protein, andto produce inositol-1,4,5-trisphosphate/diacylglycerol.

Preparation of Template DNA Fragments Encoding MBP-T43NTR-TrxA-H10

In this example, a fusion gene that is prepared by ligating a maltosebinding protein (MBP) gene at the 5′ region of NTR cDNA, and ligating athioredoxin (TrxA) gene and 10 histidine tag sequences at the 3′ regionof NTR cDNA was used. The expression vector comprising this fusion gene(pRG/III-hs-MBP-T43NTR-TrxA-H10) was reported to express and produce thefusion protein in E. coli (refer to Grisshammer, R. et al., BiochemicalSociety Transactions, vol. 27, pp. 899–903, 1999), and kindly providedby Dr. Watts (Oxford University). The plasmid vectorpRG/III-hs-MBP-T43NTR-TrxA-H10 comprising NTR cDNA was used as atemplate, and the 5′ primer:5′-GTTTAACTTTAAGAAGGAGATATACATATGAAAATAAAAACAGGTGCACG CA-3′ (SEQ IDNO:1) and 3′ primer: 5′-GCGGATAACAATTTCACACAGGAAACAGTCGACGCCAGGGTTTTCCCAGT-3′ (SEQ ID NO:2) were used for preparing thereaction mixture (25 μL) of the composition shown in Table1. The firstPCR was carried out according to the program shown in Table 2 and NTRcDNA fragment was amplified.

TABLE 1 Composition of the first PCR mixture Composition ConcentrationAmount Template plasmid 2 ng/μL   5 μL 5′ primer 2.5 μM   5 μL 3′ primer2.5 μM   5 μL dNTPs (Toyobo) 2 mM 2.5 μL 10 × Expand HF buffer(Boehringer 2.5 μL Mannheim) containing 15 mM MgCl₂ Sterilized Distilledwater 4.8 μL DNA polymerase (Boehringer Mannheim) 3500 units/mL 0.2 μL

TABLE 2 Program for the PCR STEP 1 94° C.  2 min STEP 2 94° C. 30 secSTEP 3 60° C. 30 sec STEP 4 72° C.  2 min STEP 5 GOTO 2 for 9 times STEP6 94° C. 30 sec STEP 7 60° C. 30 sec STEP 8 72° C.  2 min + 5 sec/cycleSTEP 9 GOTO 6 for 19 times STEP 10 72° C.  7 min STEP 11  4° C. forever

Next, 25 μL of the reaction mixture whose composition was shown in Table3 were prepared using the first PCR product as a template, twochemically synthesized double stranded DNA fragments, which arepartially overlapping with the template at its both terminals (5′fragment encoding T7 promoter sequence (SEQ ID No. 3) and 3′ fragmentencoding T7 terminator sequence (SEQ ID No. 4)), and 5′, 3′ primer:5′-GCCGCTGTCCTCGTTCCCAGCC-3′ (SEQ ID No. 5). The reaction mixture wasused for the second PCR according to the program shown in Table 2. As aresult, the template cDNA fragment of NTR fusion protein(MBP-T43NTR-TrxA-H10) inserted between the 5′ upstream T7 promotersequence and 3′ downstream T7 terminator sequence was obtained as shownin FIG. 1. The fusion protein comprises a maltose binding protein (MBP),a partially deleted NTR (T43NTR) at its N-terminal region, a thioredoxin(TrxA) and ten histidine tag sequences.

TABLE 3 Composition of the second PCR mixture Composition ConcentrationAmount First PCR product (template)  10 μL 5′, 3′ primer 100 μM 0.25 μL 5′ fragment 2 nM 0.625 μL  3′ fragment 2 nM 0.625 μL  dNTPs (Toyobo) 2mM 2.5 μL 10 × Expand HF buffer (Boehringer 2.5 μL Mannheim) containing15 mM MgCl₂ Sterilized Distilled water 8.3 μL DNA polymerase (Boehringer3500 units/mL 0.2 μL Mannheim)Synthesis of MBP-T43NTR-TrxA-H10 Protein by Cell-free Protein SynthesisSystem

E. coli S30 extract was prepared from E. coli BL21 strain in accordancewith the method of Zubay et al., (Ann. Rev. Geneti., 7, 267–287,(1973)). The reaction mixture for protein synthesis was constituted ofthe composition shown in the following Table 4 supplemented with 1 μL ofthe above PCR product of MBP-T43NTR-TrxA-H10 cDNA fragment and 7.2 μL ofthe above E. coli S30 extract in 30 μL of total volume. The reactionmixtures of the same composition except for addition of 0.04%, 0.4%, or1% digitonin (Wako pure chemical industries, Ltd.) in finalconcentration, or 0.01%, 0.02% or 0.2% Brij35 (SIGMA) in finalconcentration were prepared respectively. The protein synthesis reactionwas performed at 30° C. for two hours.

TABLE 4 Composition of protein synthesis reaction mixture CompositionConcentration Hepes-KOH (pH7.5) 58.0 mM Dithiothreitol 2.3 mM ATP 1.2 mMCTP, GTP, UTP Each 0.9 mM Creatine phosphate 81.0 mM Creatine kinase250.0 μg/mL Polyethylene glycol 8000(PEG8000) 4.00% (w/v) 3′, 5′ cyclicAMP(cAMP) 0.64 mM L-(−)-5-5,6,7,8-tetrahydrofolate 35.0 μg/mL E. colitotal t-RNA 170.0 μg/mL Potassium glutamate 200.0 mM Ammonium acetate27.7 mM Magnesium acetate 10.7 mM 20 kinds of amino acid Each 1.0 mM T7RNA polymerase (Toyobo) 16.0 units/μLDetection of MBP-T43NTR-TrxA-H10 Protein by Western Blotting UsingAnti-histidine Tag Antibody

After the synthesis reaction, the reaction mixture was centrifuged at12,000×g, for 20 minutes, and separated into a supernatant and aprecipitate. The precipitate was dissolved in one and a-half volume ofSDS-sample buffer. The supernatant was treated with acetone and theobtained precipitate was dissolved in one and a-half volume ofSDS-sample buffer. These samples were loaded on SDS-Polyacrylamide gelelectrophoresis using MULTIGEL 15/25 (Daiichi Pure Chemicals) as a gelmatrix. After the electrophoresis, using Semidry transfer apparatusBE-330 (BIOCRAFT Co., Ltd,) the protein samples in the gel were blottedto nitrocellulose membranes (PROTORAN BA85, pore size of 0.45 μm,Schleicher & Schuell). The nitrocellulose membranes were blocked with10-fold diluted Western Blocking Reagent (Roche) at room temperature forovernight. As a first antibody, 1000-fold diluted anti-histidine tagantibody (6×His Monoclonal Antibody, CLONTECH) was added to the membraneand incubated with the membranes at room temperature for one hour. Thenitrocellulose membranes were washed four times with TBST solution, andthen, as a second antibody, 5000-fold diluted anti-mouse IgG antibody(conjugated with horseradish peroxidase, Amersham Pharmacia Biotech) wasadded to the membranes and further incubated at room temperature for onehour. After washing the nitrocellulose membrane with TBST solution forfour times, the membranes were reacted with ECL Western BlottingDetection Reagent (Amersham Pharmacia Biotech) and examined (detected)by a lumino-image analyzer LAS-1000 plus (Fuji Photo Film Co. Ltd.Japan).

Synthesis of MBP-T43NTR-TrxA-H10 Protein by Addition of Digitonin

The result of Western blotting analysis of proteins synthesized in thepresence or absence of digitonin was shown in FIG. 2. The detected bandscorrespond to the protein that is recognized by anti-histidine tagantibody, that is, MBP-T43NTR-TrxA-H10. Incidentally, about 20 kDa bandswere detected in all samples including control samples (lanes 5 and 10)without template DNA, thus, they are considered to be proteins derivedfrom E. coli, to which the antibody binds nonspecifically. In theabsence of digitonin, the synthesized MBP-T43NTR-TrxA-H10 was insoluble(lane 1), and was not detected in the supernatant (lane 6). In thepresence of 0.04% digitonin, although most of the proteins wereinsoluble (lane 2), a small amount of the protein was detected in thesupernatant fraction (lane 7). In the presence of 0.4% or more ofdigitonin, it was found that most of MBP-T43NTR-TrxA-H10 were detectedin the supernatant (lanes 8 and 9), and little remained in the insolublefraction (lanes 3 and 4). These results indicated that a membraneprotein MBP-T43NTR-TrxA-H10 could be recovered from the supernatant bythe method of the present invention.

Synthesis of MBP-T43NTR-TrxA-H10 Protein by Addition of Brij 35

The result of Western blotting analysis of proteins synthesized in thepresence or absence of Brij35 was shown in FIG. 3. The detected bandscorrespond to the protein that is recognized by anti-histidine tagantibody, that is, MBP-T43NTR-TrxA-H10. Incidentally, about 20 kDa bandswere detected in all samples including control samples (lanes 5 and 10)without template DNA, thus, they are considered to be proteins derivedfrom E. coli, to which the antibody binds nonspecifically. In theabsence of Brij35 and in the presence of 0.01% Brij35, the synthesizedMBP-T43NTR-TrxA-H10 was insoluble (lanes 1 and 2), and was not detectedin the supernatant (lanes 6 and 7). In the presence of 0.02% Brij35,although a portion of the synthesized MBP-T43NTR-TrxA-H10 was detectedin the insoluble fraction (lane 3), most of the proteins were detectedin the supernatant fraction (lane 8). In the presence of 0.2% Brij35,only a small amount of MBP-T43NTR-TrxA-H10 was insoluble (lane 4), andalmost all amount of the protein were detected in the supernatant (lane9). These results indicated that a membrane protein MBP-T43NTR-TrxA-H10can be recovered from the supernatant in the system using Brij35 as wellas that of digitonin.

Example 2

Synthesis of a Human β2-Adrenergic Receptor (ADRB2)

ADRB2 is a member of G protein coupled receptors, a seven-spanningmembrane protein. By binding with adrenaline as a ligand, the receptorexhibits a function to activate adenylyl cyclase through acceleratory Gprotein, and to increase the concentration of intracellular cyclic AMP.The protein has already known, and the nucleotide sequence of the cDNAwas registered in GenBank (Accession No. AF022956).

Preparation of Template DNA Fragments Encoding His6-β₂

In this example, plasmid vector pFASTBacβ2-Gs comprising humanβ2-adrenergic receptor and bovine Gs fusion cDNA (obtained from Dr.Robert J. Lefkowitz (Duke University medical center) was used as atemplate, and the 5′ primer: 5′-GGTGCCACGCGGATCCATGGGGCAACCCGGGAAC-3′(SEQ ID NO:6) and 3′ primer:5′-GCGGATAACAATTTCACACAGGAAACAGTCGACTTACAGCAGTG AGTCATTTGTACTACAA-3′(SEQ ID NO:7) were used for preparing the reaction mixture (25 μL) ofthe composition shown in Table 1 by the similar manner as the method inExample 1. The first PCR was carried out according to the program shownin Table 2 and ADRB2 cDNA fragment was amplified.

Next, 25 μL of the reaction mixture whose composition was shown in Table3 were prepared using the first PCR product as a template, twochemically synthesized double stranded DNA fragments, which arepartially overlapping with the template at its both terminals (5′fragment encoding T7 promoter sequence, six histidine tag sequences andthrombin cleavage site (SEQ ID No. 8) and 3′ fragment encoding T7terminator sequence (SEQ ID No. 9)), and 5′, 3′ primer: 5′-GCCGCTGTCCTCGTTCCCAGCC-3′ (SEQ ID No. 5) by the similar manner as the method inExample 1. The reaction mixture was used for the second PCR according tothe program shown in table 2. As a result, the template cDNA fragment ofADRB2 (His6-β₂) was obtained as shown in FIG. 4. The template cDNAfragment thus obtained has T7 promoter sequence, histidine tag sequenceand thrombin cleavage site at the 5′ region thereof, and T7 terminatorsequence at the 3′ region thereof.

Synthesis of His6-β₂ Protein by Cell Free System

E. coli S30 extract was used for synthesizing His6-β₂ protein by cellfree system according to the similar manner as the method in Example 1.The reaction mixture for protein synthesis was constituted of thecomposition shown in the table 4 supplemented with 1 μL of the aboveHis6-β₂ template cDNA fragment and 7.2 μL of the above E. coli S30extract in 30 μL of total volume. The reaction mixtures of the samecomposition except for addition of 0.04%, 0.4%, or 1% digitonin (Wakopure chemical industries, Ltd.) in final concentration, 0.01%, 0.02% or0.2% Brij35 (SIGMA) in final concentration, or 0.5% β-dodecylmaltoside,NP-40, Tween20 or Triton X-100 in final concentration were preparedrespectively. In case of detecting the protein by autoradiography, 3.7kBq L-[¹⁴C]Leucine (Moravek Biochemicals) was added thereto. The proteinsynthesis reaction was performed at 30° C. for 120 minutes.

Detection of His6-β₂ Protein by Autoradiography

After the synthesis reaction, the reaction mixture was centrifuged at12,000×g, for 20 minutes, and separated into a supernatant and aprecipitate. The precipitate thus obtained was dissolved in one anda-half volume of SDS-sample buffer. The supernatant was treated withacetone and the obtained precipitate was dissolved in one and a-halfvolume of SDS-sample buffer. These samples were loaded onSDS-Polyacrylamide gel electrophoresis using MULTIGEL 15/25 (DaiichiPure Chemicals) as a gel matrix. After the electrophoresis, the gel wasdried, and the dried gel was allowed to stand with Imaging Plate(BAS-SR2040, Fuji Photo Film Co. Ltd. Japan) for 24 hours in a darkplace. Subsequently, the detection of the labeled protein was performedby using a bio-imaging analyzer BAS2500 (Fuji Photo Film Co. Ltd.Japan).

Detection of His6-β₂ Protein by Western Blotting Using Anti-histidineTag Antibody

The reaction mixture after the synthesis reaction was separated into asupernatant and a precipitate, and subjected to SDS-PAGE by the similarmanner as the method in Example 1. Subsequently, the protein samples inthe gel were blotted to nitrocellulose membranes, reacted withanti-histidine tag antibody and examined by a lumino-image analyzerLAS-1000 plus (Fuji Photo Film Co. Ltd. Japan) according to the similarmanner as the method in Example 1.

Synthesis of His6-β₂ Protein by Addition of Digitonin

The result of autoradiography of proteins synthesized in the presence orabsence of digitonin was shown in FIG. 5, and also the result of Westernblotting analysis of that was shown in FIG. 6. In FIG. 5 the labeledprotein bands synthesized in the absence of digitonin and presence of0.04% digitonin, were mainly detected in the precipitate fraction (lanes1 and 2) and little was detected in the supernatant (lane 6 and 7), andit was indicated that, in these condition, most of the proteins in thesample was insoluble. On the other hand, in case that the concentrationof digitonin was 0.4% and 1%, the labeled protein bands were mainlydetected in the supernatant (lanes 8 and 9). These results indicatedthat the synthesized proteins were prevented from being insoluble andthese proteins could be recovered from the supernatant by adding acertain concentration or more of digitonin to the cell free proteinsynthesis system.

On the other hand, in the result of Western blotting analysis byanti-histidine tag antibody (FIG. 6), about 20 kDa bands were detectedin all samples including control samples (lanes 5 and 10) withouttemplate DNA, thus, they are considered to be proteins derived from E.coli, to which the antibody binds nonspecifically. In the absence ofdigitonin, the synthesized His6-β₂ was insoluble (lane 1), and was notdetected in the supernatant (lane 6) as the result of FIG. 5. In thepresence of 0.04% digitonin, although most of the proteins wereinsoluble (lane 2), a small amount of the protein was detected in thesupernatant fraction (lane 7). In the presence of 0.4% or more ofdigitonin, most of His6-β₂ were detected in the supernatant fraction(lanes 3, 4, 8 and 9). These results indicated that a membrane proteinHis6-β₂ could be recovered from the supernatant by the method of thepresent invention.

Synthesis of His6-β₂ Protein by Addition of Brij 35

The result of Western blotting analysis of proteins synthesized in thepresence or absence of Brij35 was shown in FIG. 7. In the absence ofBrij35 and in the presence of 0.01% Brij35, the synthesized His6-β₂ wasinsoluble (lanes 1 and 2), and was not detected in the supernatant(lanes 6 and 7). In the presence of 0.02% Brij35, although a portion ofthe synthesized His6-β₂ was detected in the insoluble fraction (lane 3),most of the proteins were detected in the supernatant fraction (lane 8).In the presence of 0.2% Brij35, only a small amount of His6-β₂ wasinsoluble (lane 4), and almost all amount of the protein were detectedin the supernatant (lane 9). These results indicated that a membraneprotein His6-β₂ could be recovered from the supernatant in the systemusing Brij35 as well as that of digitonin. Incidentally, about 20 kDabands were detected in all samples including control samples (lanes 5and 10) without template DNA, thus, they are considered to be proteinsderived from E. coli, to which the antibody binds nonspecifically.

Synthesis of His6-β₂ Protein by Addition of β-dodecylmaltoside, NP-40,Tween 20 or Triton X-100

The result of autoradiography of proteins synthesized in case of addingβ-dodecylmaltoside, NP-40, Tween20 or Triton X-100 to the reactionmixture so that the final concentration thereof was 0.5% was shown inFIG. 8, and also the result of Western blotting analysis of that wasshown in FIG. 9. In both FIG. 8 and FIG. 9, the bands corresponding tomolecular weight about 46000 are speculated to be His6-β₂ protein. Theseresults indicated that, in the absence of detergent (surfactant), theprotein was detected in the precipitate fraction only (lane 1P), and wasnot detected in the supernatant fraction (lane 1S). In contrast, in thepresence of various detergents, almost same amounts of His6-β₂ proteinswere detected in the insoluble fraction (lane 2P) and the supernatantfraction (lane 2S) in case of adding 0.5% β-dodecylmaltoside.Incidentally, in case of adding the other detergents, His6-β₂ proteinwas little detected in the supernatant fraction. These results indicatedthat a membrane protein His6-β₂ could be recovered from the supernatantby adding 0.5% β-dodecylmaltoside.

Example 3 Reconstitution of a Human β-adrenergic Receptor (ADRB2)

Protein Synthesis by Dialysis Method

A template cDNA fragment for expression of human β2-adrenergic receptor(ADRB2) was prepared according to the same manner as Example 2, andfurther cloned into plasmid pCR2.1-TOPO by using TOPO TA Cloning Kit(Invitrogen Co.) to use as a template. Next, His6-β₂ protein wassynthesized by cell free protein synthesis system using E. coli S30extract according to the same method as Example 1. However, in contrastto Example 1 and 2, the protein synthesis was performed by the dialysismethod using an internal reaction mixture (20 mL) and an externalreaction mixture (200 mL), whose compositions were shown in Table 5. Theinternal mixture was dispensed with 5 mL each in four dialysis membranes(DispoDialyzer, Spectra/Por, fraction molecular weight 50000), and wassuspended in the external reaction mixture for synthesizing protein at30° C. for 16 hours.

TABLE 5 Concentration Composition Internal dialysate External dialysateHepes-KOH (pH7.5) 58.0 mM 58.0 mM Dithiothreitol 2.3 mM 2.3 mM ATP 1.2mM 1.2 mM CTP, GTP, UTP Each 0.9 mM Each 0.9 mM Creatine phosphate 81.0mM 81.0 mM Creatine kinase 250.0 μg/mL 250.0 μg/mL Polyethylene glycol4.00% (w/v) 4.00% (w/v) 8000(PEG8000) cyclic AMP(cAMP) 0.64 mM 0.64 mML-(−)- 35.0 μg/mL 35.0 μg/mL 5-5,6,7,8-tetrahydrofolate E. coli totalt-RNA 170.0 μg/mL 170.0 μg/mL Potassium glutamate 200.0 mM 200.0 mMAmmonium acetate 27.7 mM 27.7 mM Magnesium acetate 10.7 mM 10.7 mMSodium azide 1.5 mM 1.5 mM 20 kinds of amino acid Each 1.5 mM Each 1.5mM T7 RNA polymerase 16.0 units/μL — (Toyobo) E. coli S30 extract 7.2μL/30 μL of — total volume Plasmid DNA 1.0 μL/30 μL of — total volumeDigitonin 0.4% 0.4% Lipid solution 0.5 μL/30 μL of — total volumePotassium acetate — 3.0 mM Tris-acetate — 4.2 mMDialysis

After the synthesis reaction, the above dialysis membranes weretransferred into phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂,HPO4, 2 mM KH₂PO₄), containing 1% CARBIOSORB (Carbiochem), forperforming dialysis at 4° C. for 8 hours while replacing the bufferevery two to three hours.

Solubilization

After the dialysis, the internal dialysate was recovered, andultracentrifuged at 100,000×g for one hour at 4° C. The precipitatefraction thus obtained was suspended in 15 mL of the above phosphatebuffer, and supplemented by drop with 10% β-dodecylmaltoside(NacalaiTesque Inc.) to adjust 1% of the final concentration, and thenwas solubilized at 4° C. for 2 hours. The solubilized solution wastransferred into dialysis membrane tube (Spectra/Por, fraction molecularweight 10,000), and was dialyzed in the above phosphate buffer at 4° C.for 8 hours.

Purification

The solubilized solution after dialysis was ultracentrifuged at 4° C.,100000×g for one hour. The supernatant fraction thus obtained (15 mL)was added with Ni—NTA agarose (QIAGEN) (wet volume 2 mL) which waspre-equilibrated with buffer A (20 mM phosphate buffer (pH7.4), 500 mMNaCl, 10 mM imidazole, 0.05% β-dodecylmaltoside), and then mixed gentlyat 4° C. for 3 hours. Next, the Ni—NTA agarose was packed to a columnfor removing the extra buffer therefrom. After washing the column with20 mL of buffer A, the protein was eluted with 5 mL of buffer B (20 mMphosphate buffer (pH7.4), 500 mM NaCl, 300 mM imidazole, 0.05%β-dodecylmaltoside).

Desalting

5 mL of the protein eluate was concentrated by VIVASPIN (Sartorius K.K., fraction molecular weight 10000) up to 2.5 ml. 2.5 mL of theconcentrate was added to PD-10 desalting column (Amersham Pharmacia)pre-equilibrated with phosphate buffer, followed by adding phosphatebuffer to the column to recover an eluate fraction of protein (3.5 mL).

Reconstitution

3.5 mL of the protein eluate was concentrated by VIVASPIN (fractionmolecular weight 10000) up to 1 ml. To this 1 mL concentrate, 0.01%β-dodecylmaltoside in final concentration and 6.25 μL of mixed lipidsolution was added, and the mixture was transferred to dialysis membranetube (Spectra/Por, fraction molecular weight 50000) for dialyzing inphosphate buffer containing 1% CARBIOSORB at 4° C. for 8 hours.

Detection of the Reconstituted His6-β₂Protein

The purity of His6-β₂ protein reconstituted in the presence of detergentand lipid according to the above method was analyzed by SDS-PAGE andWestern blotting by the same method of Examples 1 and 2. FIG. 10 showsthe results of subjecting samples in each step of purification by Ni-NTAagarose and PD-10desalting column to (a) silver staining, and (b) thewestern blotting analysis using anti β2 AR antibody, after SDS-PAGE Bothresults of (a) and (b) indicates that His6-β₂ protein (molecular weightabout 46 kDa) was purified.

Binding Assay

The dialyzed protein solution reconstituted by the above methods wasultracentrifuged at 4° C., 100,000×g for one hour. The obtainedprecipitate fraction (the reconstituted membrane fraction) was suspendedin 100 to 200 μL of incubation buffer (75 mM Tris-HCI (pH7.4), 12.5 mMMgCl₂, 2 mM EDTA). This reconstituted membrane fraction was allowed tostand in the presence of 0 to 100 μM of Alprenolol (Sigma Co.) at 30° C.for 30 minutes. Subsequently, [³H]Dihidroalprenolol was added thereto asto make up 10 μM of the final concentration, and further reacted at 30°C. for one hour.

96 well Unifilter GF/C (Whatman) was prepared, and previously washed twotimes with 200 μL of 0.3% polyethyleneimine, and subsequently nine timeswith 200 μL of 50 mM Tris-HCl (pH7.4). To this 96 well Unifilter, theabove reaction mixture was added, and subsequently washed seven timeswith incubation buffer. Then, the 96 well Unifilter was dried, and toeach well of the dried Unifilter, 50 μL of MicroScint-0 (Packard) wasadded. This Unifilter was allowed to stand in a dark place for 10minutes. The radioactivities derived from [³H]Dihidroalprenolol ofrespective wells of this Unifilter were determined by using TOPCOUNT(Packard). The incorporated amount of [³H]Dihidroalprenolol in thepresence of various concentration of Alprenolol (Binding Curve) wasshown in FIG. 11. In FIG. 11, the human β2AR (Sf9) (Lot No. UHW-1098F)used as a control was purchased from RBI. In FIG. 11, a molarconcentration of added Alprenolol was plotted on the x-axis in a logscale, and an incorporated ratio of radioactivity in the presence ofrespective concentrations of Alprenolol in case of taking aradioactivity determined in the absence of Alprenolol as 100% wasplotted on y-axis. From FIG. 11, it is found that, the efficiency ofincorporation of [³H]Dihidroalprenolol into His6-β₂ proteinreconstituted by the method of the present invention, decreases fromabout 80% to about 10% according as the concentration of Alprenololincreases from 10⁻⁶M to 10⁻⁴M, that is, the protein binds to the addednon-labeled Alprenolol.

INDUSTRIAL APPLICABILITY

According to the present invention, it is capable of obtaining aconstant amount of protein, especially membrane protein, without loss ofactivity of the protein, and providing a useful method for the researchof structure and function of the protein. Through the development ofdrugs to control the function of these membrane protein, in particular,a receptor, channel protein and transporter, a variety of applicationsfor diagnostics and treatment are expected.

1. A method for producing an eukaryotic membrane protein using acell-free protein synthesis system comprising a detergent to synthesizethe protein without aggregation, wherein the detergent is digitonin inan amount of 0.4 to 1.5% by volume or polyoxyethylene 23 lauryl ether inan amount of 0.02 to 0.2% by volume, or a mixture of digitonin in anamount of 0.4 to 1.5% by volume and polyoxyethylene 23 lauryl ether inan amount of 0.02 to 0.2% by volume.
 2. The method of claim 1, whereinsaid membrane protein is a protein that comprises a hydrophobic region,or said membrane protein is a portion of said membrane proteincomprising a hydrophobic region.
 3. A method for reconstituting aprotein produced in a cell-free protein synthesis system comprising anucleic acid template coding for at least a portion of a membraneprotein comprising a hydrophobic region, a detergent that is digitoninin an amount of 0.4 to 1.5% by volume or polyoxyethylene 23 lauryl etherin an amount of 0.02 to 0.2% by volume, or a mixture of digitonin in anamount of 0.4 to 1.5% by volume and polyoxyethylene 23 lauryl ether inan amount of 0.02 to 0.2% by volume, and a lipid, wherein said proteinis reconstituted in a lipid bilayer by decreasing the concentration ofsaid detergent in said system simultaneously with the protein synthesisor after a period therefrom.
 4. The method of claim 3, wherein the stepof decreasing the concentration of said detergent is performed by anyone or more methods selected from the group consisting of dialysis,dilution, filtration, centrifugation and addition of an adsorbent tosaid detergent.
 5. A method for producing a membrane protein comprisingthe steps of: (a) synthesizing the protein in a cell-free proteinsynthesis system comprising a cell extract, a nucleic acid templatecoding for said protein, a detergent that is digitonin in an amount of0.4 to 1.5% by volume or polyoxyethylene 23 lauryl ether in an amount of0.02 to 0.2% by volume, or a mixture of digitonin in an amount of 0.4 to1.5% by volume or polyoxyethylene 23 lauryl ether in an amount of 0.02to 0.2% by volume and a lipid; and (b) decreasing the concentration ofsaid detergent in the reaction mixture simultaneously with the proteinsynthesis or after a period therefrom, wherein the synthesized proteinhas at least a part of its biological activity.
 6. The method of claim5, wherein said membrane protein is a protein selected from the groupconsisting of a receptor, a channel protein, a transporter, and amembrane-bound enzyme.
 7. The method of claim 3, wherein said cell-freeprotein synthesis system comprises a bacterial cell extract.
 8. Themethod of claim 5, wherein said cell extract is a bacterial cellextract.
 9. The method of claim 1, wherein said eukaryotic membraneprotein is a G protein coupled receptor.
 10. The method of claim 3, inwhich the protein is a G protein coupled receptor.
 11. The method ofclaim 5, in which the protein is a G protein coupled receptor.